1. Field of the Invention
The present invention relates to a method for sample preparation efficiently provided for a DNA sequencing process, which basic technology utilizes an enzymatic extension reaction of complementary strand. The present invention also relates to a reagents kit for use in the DNA sequencing process.
2. Description of the Related Art
Focusing on human genome analyses, a high throughput and highly efficient DNA sequencing technology is required. DNA sequencing starts from the preparation of DNA library which covers all DNAs, by making clones having a length of 10 Kbp to 100 Kbp from the DNAs present in a gene. DNA sequencing of clones thus produced has been conventionally relied upon and roughly classified into three techniques, i.e., shotgun, primer-walking and nested-deletion techniques.
The shotgun technique involves digesting a sample DNA at random by sonication, preparing DNA fragments by subcloning and sequencing each fragment, whereby overlaps of the sequenced fragments are used to determine the full-length base sequence. Details of the shotgun technique are described in, e.g., T. Maniatis et al.: Molecular Cloning, Cold Spring Harbor Laboratory Press, 13, 21-33 (1989).
According to the primer-walking technique, sequences of DNA fragments to be sequenced are sequentially determined one by one from the terminus thereof. That is, in the primer-walking technique, one end of a sample DNA is first sequenced. Next, sequences for a primer contiguous to the following region are selected from the thus determined base sequences. DNA sequencing again starts from the selected sequences. Technical details on the primer walking technique are described in, e.g., T. Maniatis et al.: Molecular Cloning, Cold Spring Harbor Laboratory Press, 13, 14-20 (1989).
In the nested-deletion technique, fragments from a sample DNA are subjected to enzymatic digestion to prepare fragments with slightly different sizes. After one end of these fragments is trued up, the fragments are sequenced from another end in the order of a longer length. Details of the nested-deletion technique are found in, e.g., T. Maniatis et al.: Molecular Cloning, Cold Spring Harbor Laboratory Press, 13, 34-41 (1989).
In the shotgun method described above, what portion of the sample DNA corresponds to DNA extracted by subclone is unknown until the full-length base sequence is determined. For this reason, it is necessary to analyze the DNA length longer by 10 to 20 times than the length of a DNA strand to be actually sequenced. Therefore, intensive time and labors are required, resulting in serious obstacles. Turning to the primer walking technique mentioned above, this technique provides more efficient DNA sequencing but involves disadvantages, since successive operations for sequencing is time-consuming and a primer must be prepared every time when sequencing is done.
According to the nested-deletion technique mentioned above, new information on a sequence corresponding to the difference in length between fragments is always obtained. The nested-deletion technique is thus efficient, because DNA sequencing is performed sequentially from one end, as in the primer-walking method. Furthermore, the thus prepared DNA fragment is inserted into a plasmid for subcloning and the subclone is then sequenced. Accordingly, a priming site is obtained from the base sequence of plasmid, meaning that there is no need to prepare a primer for DNA sequencing every time. In view of these characteristics of the nested-deletion method, it appears that this method may overcome the problems encountered in the shotgun and primer walking techniques. From a practical viewpoint, however, time-consuming operations for selecting and arranging samples convenient for DNA sequencing are required after all. This is a key how to meet the requirement. In addition, purification by subcloning that is commonly performed in the shotgun and nested-deletion methods is such a complicated operation that is a serious problem to be solved.
In order to solve the problems of complicated operations required for DNA sequencing as recognized in the three techniques hitherto applied, various attempts have been made. In particular, direct sequencing of DNA fragments in a mixture obtained from a sample DNA digested with a restriction enzyme is a promising method. This technique is called a fragment-walking method, which is briefly explained below and a more detailed explanation is found in, e.g., K. Okano et al., Gene, 176: 231-235 (1996), "Fragment walking for large DNA sequencing by using a library as small as 16 primers". A sample DNA is digested with a restriction enzyme. Thereafter an oligomer having a known sequence is ligated with the DNA fragment at the terminus thereof through a ligation reaction to recognize each DNA fragment in the fragments mixture. Sequencing reaction is then conducted using a set of primers which can discriminate a complementary base sequence to at least a part of the ligated oligomer and the restriction enzyme recognition sequence, and an unknown one-base to four-bases sequence adjacent to the recognition sequence. The primer set includes, for example, 16 combinations of all possible bases in the case of an unknown base sequence having variable two base sequence. Where a small number of DNA fragments are contained in a DNA fragment mixture and one DNA fragment forms a complete base pairing with one primer, the base sequence of each DNA fragment can be determined from the mixture, respectively, using the set of primers described above. Where one primer hybridizes to at least two DNA fragments, the above procedure is performed, e.g., after separation of DNA fragments depending upon size. After the base sequence of each DNA fragment has been determined, the base sequences of the respective DNA fragments are reconstructed to obtain the overall base sequence. In order to obtain the overall base sequence, there is employed a method in which fragments are walked over to determine the DNA sequence of the overlapping portion, using as a template for sequencing each fragment and a full-length intact sample DNA before a digestion reaction with any restriction enzyme. Alternatively, there is employed another method in the number of restriction enzymes used initially is increased and the overall DNA sequence is determined by making use of the overlapping base sequence between the fragments.
As explained above, those conventional techniques are all somehow disadvantageous. That is, the shotgun technique requires repeated reading of the same base sequence and is thus inefficient due to high redundancy. For reducing redundancy, the primer-walking technique and the nested-deletion technique have been proposed. However, the primer-walking technique involves disadvantages that prior to DNA sequencing, primers must be synthesized every time or it is necessary to prepare a library including a huge number of primers and it takes much time for DNA sequencing in a series. Turning to the nested-deletion technique, this method is disadvantageous in that a sample convenient for DNA sequencing is prepared only with difficulty. Additional problems encountered with the shotgun and nested-deletion techniques are seen in much time and labors required for cloning using a culture step that is automated also only with difficulty. Contrary to the two techniques above, the fragment-walking method is advantageous in that this technique does not require such cloning and provides low redundancy but is reduced but where the digested fragments have long DNA sequences, difficulties occur in their connection.